The long-term objective of this project is to learn more about how chromatin structure regulates transcriptional gene silencing in eukaryotes. The basic mechanisms used to generate inactive or silent chromatin are largely conserved in eukaryotic organisms ranging from yeast to humans. Our studies focus on understanding the roles of chromatin-associated factors, such as histones, histone-modifying enzymes, and silencing proteins, in regulating the silencing of RNA polymerase II-transcribed genes located in the ribosomal DNA locus (rDNA) of the budding yeast Saccharomyces cerevisiae. The experiments in this proposal will use a combination of genetic, molecular, and biochemical approaches to address how histones and silencing factors affect gene silencing and chromatin structure in the rDNA. First, studies to characterize the histone H3 methyltransferase complex, COMPASS, will be performed. Gene silencing is one of the only cases in S. cerevisiae, where H3 methylation has a clear effect on gene expression. The proposed experiments will investigate the role of COMPASS proteins in rDNA silencing, determine how rDNA chromatin is altered by methylation of histone H3, and address the possibility that methylated histone H3 recruits silencing factors to the rDNA. Mutagenesis studies focusing on the catalytic subunit of COMPASS coupled with in vivo and in vitro assays for COMPASS function will advance the molecular characterization of this evolutionarily conserved catalytic domain. Second, the histone composition of rDNA chromatin will be analyzed using molecular and genetic approaches. These studies will not only shed light on how histones and silencing factors function in gene silencing in S. cerevisiae, they will provide information about how related proteins function in humans. Furthermore, gene silencing is critical for normal growth and development. When the processes that regulate gene silencing are disrupted in mammalian cells, inappropriate gene expression and excessive recombination can lead to diseases, including cancer and leukemia. Our studies investigating the mechanisms that cells use to control gene silencing will provide insight into how changes in regulatory pathways can lead to malignancy and will contribute to the identification of targets for future therapeutics.